1. FIELD OF THE INVENTION
The present invention relates to novel phenylazonapthyl-N-acetyl-.beta.-D-glucosaminide derivatives, reagents for determination of N-acetyl-.beta.-D-glucosaminidase activity and a method for determination of N-acetyl-.beta.-D-glucosaminidase activity.
N-Acetyl-.beta.-D-glucosaminidase is one of glycolytic enzymes and widely distributed in vital tissues, especially rich in the kidney. A high N-acetyl-.beta.-D-glucosaminidase activity is noted particularly around the proximal convoluted kidney tubule.
Determination of N-acetyl-.beta.-D-glucosaminidase activity is of clinically great significance as giving useful information for early diagnosis of denial reaction after renal transplantation, diagnosis and course observation of various diseases such as acute renal insufficiency, glomerulonephritis, etc., renal toxicity with drug, and the like.
For determination of N-acetyl-.beta.-D-glucosaminidase (hereafter often simply referred to as NAG) activity, it is conventional to react NAG on a substrate obtained by binding p-nitrophenol to N-acetyl-.beta.-D-glucosamine at the reducing end thereof and perform colorimetry of p-nitrophenol released [Methods Engymol., 28. 702 (1972) and Biochem. J., 78. 106 (1961)].
In this method, however, it is necessary to determine urine blank. In addition, a molecular extinction coefficient of a colored substance is relatively small so that the method involves a defect that a reaction should be carried out over long periods of time to enhance analysis sensitivity.
A method using a substrate obtained by binding 4-methylumbelliferone to N-acetylglucosamine [Clin. Chim. Acta, 24. 189 (1969)]is also used. However, the methods encounter problems that since the reaction is carried out at a substrate concentration lower than Km value due to poor solubility of the substrate, the method tends to be affected by interferents in urine and urine blank must be measured as in the method using p-nitrophenol.
As an improved substrate for requiring no blank test, there is known m-cresolsulfophthalyl N-acetyl-.beta.-D-glucosaminide (Japanese Patent Application KOKAI (Laid-Open) No. 58-994 and Japanese Patent Application KOKOKU (published for purpose of opposition) No. 63-7196). However, since these methods are directed to endpoint assay which comprises adding a reagent for terminating the reaction and forming a color in an alkaline region, the methods involve drawbacks that the assay should be performed using two reagents and it takes a long time and therefore, type of machines used for automated operations is limited, and the like.
In order to eliminate the drawbacks in the known methods, there has been proposed a rate assay for determining a difference in absorbance of NAG activity with passage of time in a definite time period during which the enzyme reaction proceeds. As substrates applicable to such rate assay, there are known 2-chloro-4-nitrophenyl-N-acetyl-.beta.-D-glucosaminide 4-chloro-2-nitrophenyl-N-acetyl-.beta.-D-glucosaminide (Japanese Patent Application Laid-Open No. 61-112092). However, solubility of the substrate is poor and the reaction is carried out at a substrate concentration lower than Km value. Since the method tends to be affected by interferents in urine and the assay is made at a wavelength of 405 nm, it is troublesome in that urine blank must be measured as in the method using p-nitrophenol.
In colorimetry, urine blank is measured to eliminate interference caused mainly by billirubin and hemoglobin. In order to omit the urine blank measurement, it is necessary that a wavelength for the measurement be set in a region longer than 540 nm which is free of interference by these substances.
As a result of various investigations to solve these problems, the present inventors have succeeded in accurately determining NAG activity in a sample in a simple manner using a solution containing a phenylazonaphthyl-N-acetyl-.beta.-D-glucosaminide derivative as substrate.